Safe and effective vectored virus vaccines have great potential for improving global health. In the developing world, respiratory and gastrointestinal infections are major causes of death in young children and vaccine platforms are needed that can safely deliver multiple vaccine antigens in a single dose. The over-riding goal of this proposal is to develop the human coronavirus NL63 strain as a heterologous vaccine vector, especially for use in the developing world, which has poor access to vaccines or intellectual property dedicated toward improving human health. We have chosen the development and testing of NL63 vaccine vectors because they deliver respiratory pathogen antigens to the appropriate mucosal epithelial cells lining the upper and lower airways, induce robust humoral, mucosal and cellular immune responses, the genome organization allows for simultaneous and regulated expression of multiple foreign proteins, and reversion and recombination proof technologies have been developed to make coronavirus vectors safe and replication deficient. Using our recently developed molecular clone of the human coronavirus strain, NL63, we will use computer design, synthetic genomics and reverse genetics to engineer safe, heterologous vaccine vectors for the improvement of Global Public Health. The specific aims of this proposal are designed to build candidate vectors and to develop an animal model infrastructure for vaccine testing and development. The impact of these studies is potentially high, leading to new vaccine technologies for the improvement of global health. Public Health Relevance: In the developing world, respiratory and gastrointestinal infections are major causes of death in young children and vaccine platforms are needed that can safely deliver multiple vaccine antigens in a single dose. The over-riding goal of this proposal is to develop the human coronavirus NL63 strain as a heterologous vaccine vector, especially for use in the developing world, which has poor access to vaccines or intellectual property dedicated toward improving human health. We have chosen the development and testing of NL63 vaccine vectors because they deliver respiratory pathogen antigens to the appropriate mucosal epithelial cells lining the upper and lower airways, induce robust humoral, mucosal and cellular immune responses, the genome organization allows for simultaneous and regulated expression of multiple foreign proteins, and reversion and recombination proof technologies have been developed to make coronavirus vectors safe and replication deficient.